Lethal pulmonary infection with Francisella novicida causes depletion of alphabeta T cells from lungs

Abstract

Respiratory Francisella infections induce a delayed innate immune response followed by a severe sepsis like condition. In this study, mice infected intranasally with Francisella novicida showed a depletion of alphabeta T cells in lungs while exhibiting large accumulations of other leukocytes correlating with disease severity. The depleted T cells were predominantly CD4(+). The alphabeta T cells in infected mice showed significantly higher levels of Annexin V binding than those in mock control mice suggesting increased apoptosis of T cells. These results suggest that lack of transition from an innate to adaptive host response is associated with lethality of respiratory tularemia.

Figure 1

Intranasal infection with F. novicida results in depletion of αβ T cells from lungs. Lungs from mock control and mice infected intranasally with F.n. were processed for in-situ IF staining or for FACS analysis as described in Materials and Methods. (A, upper panel) Lungs from infected mice showed reduced number of αβ T cells (red) while the number of CD11b+ cells (red) was substantially higher in infected lungs in comparison to mock lungs at 72 h p.i. Nuclei (blue) are stained with 4′6′ diamidino-2-phenylindol-dilactate (DAPI). Images are from one representative experiment of three performed (n= 3–4 mice per group for each experiment). Magnification X 200. Lower panel shows FACS analysis of lung cells from mock control or mice infected with F.n. at 72 h p.i. The cells were stained for αβ T cells and CD11b+ cells as described in Methods. Appropriate isotype matched negative controls were used to set the gates. The positively stained cells were expressed as % of total lung cells. An average of % positive cells from three mice per group of one representative experiment is shown. The total number of lung cells (× 10⁶) in mock mice was 7.9 ± 1.9 and in F. novicida infected mice was 10.2 ± 1.2 in this representative experiment. Experiment was repeated three times with similar results. ***, p < 0.0005. (B). Upper panel shows FACS analysis of lung cells from mock control or infected mice at 72 h p.i. stained simultaneously for CD4+ and TCR βor CD8+ and TCR β markers. The numbers on upper right box of each image depict % of double positive cells. Lower Panel shows graphical representation of these results. The positively stained cells were expressed as % of total lung cells. An average of % positive cells from three mice per group is shown. Experiment was repeated two times with similar results. ***, p < 0.0005.

Figure 2

αβ T cells in lungs undergo apoptosis during pulmonary F.n. infection. (A) Gating scheme for identification of apoptotic αβ T cells in mice lungs is shown. The lung cells from mock control or infected mice at 72 h p.i. were stained simultaneously with APC conjugated rat anti-mouse TCR β antibody for αβ T cells, FITC conjugated Annexin V and propidium iodide. Lymphocyte population in lungs (P1) was gated to identify αβ T cells (P2). P2 was then gated to identify propidium iodide positive necrotic cells (P3). P3 was then excluded to identify Annexin V positive apoptotic αβ T cell population. B, Upper panel shows a representative image of FACS analysis of apoptotic αβ T cells in mock control and F.n. infected mice lungs at 72 h p.i. The positively stained cells are expressed as % of αβ T cell population in lower panel. An average of % positive cells from one representative experiment with three mice per group is shown. The experiment was repeated three times with similar results. *, p <0.05; **, p < 0.005, ***, p < 0.0005.